Abstract:

Methods for identifying MarR family inhibiting compounds are described.
The methods include the use of computer aided rational based drug design
programs and three dimensional structures of MarR family polypeptides.

Claims:

1.-9. (canceled)

10. A method of identifying a MarR family modulating compound
comprising:determining the structure of a MarR family polypeptide using
the structure of MarR, such that a MarR family modulating compound is
identified.

11. The method of claim 10, wherein said method further comprises
identifying a candidate MarR family modulating compound by performing
rational drug design based on the structure.

12. The method of claim 11, wherein said method further comprises
contacting the candidate MarR family modulating compound with a MarR
family peptide, and a nucleic acid molecule.

13. The method of claim 12, wherein said method further comprises
measuring the binding affinity of the MarR family polypeptide peptide
with the nucleic acid molecule.

37. The method of claim 22, wherein said MarR modulating compound is of
the formula: ##STR00005## whereinY is a substituted or unsubstituted
cyclic or bicyclic moiety, and pharmaceutically acceptable salts and
esters thereof.

38.-40. (canceled)

41. The method of claim 37, wherein said MarR modulating compound is
selected from the group consisting of: ##STR00006## ##STR00007##
##STR00008##

42. A method for inhibiting expression of MarA, comprising contacting MarR
with a MarR modulating compound, wherein said MarR modulating compound is
of the formula (I):X--Y--Z (I)whereinX is an interacting moiety;Y is a
hydrophobic moiety; andZ is a polar moiety.

[0003]MarR is a regulator of multiple antibiotic resistance in Escherichia
coli. It is the prototypic member of a family of regulatory proteins
found in the Bacteria and the Archae that play important roles in the
development of antibiotic resistance, a global health problem. In the
absence of an appropriate stimulus, MarR negatively regulates expression
of the marRAB operon (Cohen, S. P., et al. 1993. J. Bacteriol. 175:
1484-1492.; Martin, R. G. and Rosner, J. L. 1995. Proc. Natl. Acad. Sci.
92: 5456-5460; Seoane, A. S. and Levy, S. B. 1995. J. Bacteriol. 177:
3414-3419, 1995). DNA footprinting experiments suggest that MarR
dimerizes at two locations, sites I and II, within the mar operator
(marO) (Martin and Rosner, 1995, supra). Site I is positioned among the
-35 and -10 hexamers and site II spans the putative MarR ribosome binding
site (reviewed in Alekshun, M. N. and Levy, S. B. 1997. Antimicrob.
Agents Chemother. 10: 2067-2075).

[0004]MarR is a member of a newly recognized family of regulatory proteins
(Alekshun, M. N. and Levy, S. B. 1997. Antimicrob. Agents Chemother. 10:
2067-2075. Sulavik, M. C., et al. 1995. Mol. Med. 1: 436-446) and many
functional homologues have been identified in a variety of important
human pathogens and have been found to regulate a variety of different
processes. For example, some MarR homologues have been found to control
expression of multiple antibiotic resistance operons, some regulate
tissue-specific adhesive properties, some control expression of a cryptic
hemolysin, some regulate protease production, and some regulate
sporulation. Proteins of the MarR family control an assortment of
biological functions including resistance to multiple antibiotics,
organic solvents, household disinfectants, and oxidative stress agents,
collectively termed the multiple antibiotic resistance (Mar) phenotype
(Alekshun, M. N. & Levy, S. B. Trends Microbiol. 7, 410-413 (1999)).
These proteins also regulate the synthesis of pathogenic factors in
microbes that infect humans and plants (Miller, P. F. & Sulavik, M. C.
Mol. Microbiol. 21, 441-448 (1996)). Insight into the three dimensional
structure of MarR family proteins would be of great value in designing
drugs that interact with this family of proteins and modulate MarR
function, for example, antibiotic resistance and virulence.

SUMMARY OF THE INVENTION

[0005]The instant invention advances the prior art by providing the
crystal structure of a MarR family polypeptide, MarR. The crystal
structure of MarR provides the three-dimensional structure, as well as
the shape and electronic properties of its active sites. It can be used
in a comprehensive rational drug design program to develop novel
chemotherapeutics targeted toward the MarR/MarA transcription system. The
atomic coordinates of a MarR crystal structure cocrystallized with and
without salicylate are given in FIG. 1 and FIG. 2, respectively.

[0006]In one embodiment, the invention pertains, at least in part, to
methods for identifying a MarR family modulating compound. The method
includes selecting a candidate MarR family modulating compound by
performing rational drug design with the set of atomic coordinates in
FIG. 1 or 2. The method may further include contacting the candidate MarR
family modulating compound with the MarR family polypeptide, and
determining the ability of the candidate MarR family modulating compound
to modulate the MarR family polypeptide. The invention also pertains to
compounds identified by these methods and methods of using the compounds
to modulate MarR family polypeptides.

[0007]In another embodiment, the invention pertains, at least in part, to
methods for identifying a MarR family modulating compound. The methods
include determining the structure of a MarR family polypeptide using the
structure of MarR and identifying a candidate MarR family modulating
compound by performing rational drug design based on the structure. The
method may further include the steps of contacting the candidate MarR
family modulating compound with a MarR family peptide, and a nucleic acid
molecule, and measuring the binding affinity of the MarR family
polypeptide peptide with the nucleic acid molecule. The invention also
pertains to compounds identified by these methods and methods of using
the compounds to modulate polypeptides.

[0008]In yet another embodiment, the invention pertains, at least in part,
to a method for identifying a MarR modulating compound. The method
includes obtaining a set of atomic coordinates defining the
three-dimensional structure of MarR and selecting a candidate MarR
modulating compound by performing rational drug design with the three
dimensional structure of MarR. The method may further include the steps
of contacting the candidate MarR modulating compound with MarR, measuring
the ability of the candidate MarR modulating compound to modulate the
activity of MarR. The invention also pertains to compounds identified by
these methods and methods for modulating MarR using the compounds of the
invention.

[0009]In another embodiment, the invention pertains, at least in part, to
a MarR modulating compound of the formula (I):

X--Y--Z (I)

wherein X is an interacting moiety; Y is a hydrophobic moiety; and Z is a
polar moiety.

[0010]The invention also pertains, at least in part, to methods for
inhibiting expression of MarA, by contacting MarR with a MarR modulating
compound of formula (I).

[0011]In yet another embodiment, the invention also pertains to methods
for decreasing multidrug resistance in a microbe. The method includes
contacting the microbe with a MarR modulating compound of formula (I).

BRIEF DESCRIPTION OF THE DRAWINGS

[0012]The file of this patent contains at least one drawing executed in
color. Copies of this patent with color drawing(s) will be provided by
the Patent and Trademark Office upon request and payment of the necessary
fee.

[0013]FIG. 1 shows the atomic coordinates of the MarR-salicylate
co-crystal.

[0014]FIG. 2 shows the atomic coordinates of the MarR crystal without
salicylate.

[0015]FIG. 3 shows the sequence alignment of MarR with representative
members of the MarR family.

[0016]FIG. 4 is a ribbon representation of the salicylate containing MarR
dimer with the two-fold axis near vertical. There are two salicylate
molecules per monomer and each is represented by a stick model.

[0017]FIG. 5 is a representation of the N-/C-terminal domain represented
by a surface (red) around the van der Waals radii of the side chain atoms
only of the hydrophobic core residues. The main chain and other residues
of the domain are shown in yellow for one subunit and blue for the other.
Helices leading to and from the domain are shown in ribbon
representation.

[0018]FIG. 6 is an electrostatic surface representation of the MarR dimer.

[0019]FIG. 7 is a Cα trace of a MarR subunit in stereo
representation.

[0020]FIG. 8 is a diagram which shows interactions between the DNA-binding
domains of the dimer in the region of the Arg 73-Asp 67' salt bridges.
The stereo view is coincident with the 2-fold rotation axis of the dimer.
Electron density shown is a 2FO--FC map contoured at 1σ.

[0021]FIG. 9 shows a ribbon representation of the MarR dimer with the
two-fold axis near vertical.

[0025]FIG. 13A represents the SCRs of MarR derived from the MarR crystal
structure. The basic framework of SlyA is shown in FIG. 13B.

[0026]FIG. 14 shows a C.sub.α-tube representations of MarR from the
crystal structure and its homology with a model of SlyA.

DETAILED DESCRIPTION OF THE INVENTION

[0027]Chemotherapeutic intervention for the treatment and prevention of
disease is predicated upon the ability of small molecules (drugs) to
infiltrate a biological system and to interact with the components of the
biological system (e.g. proteins, RNA, DNA, membranes, etc.) in a manner
that modulates their normal function. Rational drug design attempts to
formulate drug design hypotheses that specify and optimize the physical
contacts between the drug and target. Koshland has used a lock and key
analogy to characterize drug-target interactions; a specific "key" (drug)
interacts only with its respective molecular "lock" (target) (Koshland,
D. E., Jr. Angew. Chem. 1994, 106, 2468-2472). This model asserts that an
appropriate degree of shape and electronic complimentarily between the
drug and target must occur to produce productive drug-target
interactions--those that cause a desired pharmacological response. The
specific location on the "lock" or target is referred to as the active or
catalytic site. The three dimensional shape and electronic properties of
the active site form the basis for rational drug design and provides
information toward the systematic chemical modifications of potential
drugs.

[0028]In one embodiment, the invention pertains to methods for identifying
MarR family modulating compounds using the three-dimensional structure of
a MarR family polypeptide. The method includes selecting a candidate MarR
family modulating compound by performing rational drug design with the
atomic coordinates of a MarR family polypeptide. The method may also
include contacting the candidate MarR family modulating compound with
MarR family polypeptide; and determining the ability of said candidate
MarR family modulating compound to a modulate MarR family polypeptide. In
one embodiment, the MarR family polypeptide is MarR. The atomic
coordinates of MarR in the presence and absence of salicylate are given
in FIGS. 1 and 2, respectively.

MarR Family Polypeptides and Nucleic Acid Molecules

[0029]The term "MarR family polypeptide" includes molecules related to
MarR, e.g., having certain shared structural and functional features.
MarR family polypeptides also include those which are structural homologs
of MarR. The structural homologs include those having a crystallized form
which are structurally similar to that of crystallized MarR. Generally,
it is believed that there is a strong relationship between the tertiary
structure of a protein and its function within the biological system.
Furthermore, it is known that a protein's overall tertiary structure is
related to its primary amino acid sequence. Therefore, it has been
demonstrated that proteins with similar amino acid make up and sequence
will possess similar overall structure and will likely share similar
function. MarR family members, in addition to having similarity to MarR,
may bind to DNA and regulate transcription. While some MarR family
members negatively control transcription (e.g., MarR), others have
positive/activator functions (e.g., SlyA, BadR, NhhD, and MexR). MarR
family polypeptides comprise DNA and protein binding domains. In
addition, MarR family polypeptides can interact with a variety of
structurally unrelated compounds that regulate their activity.

[0030]Exemplary MarR family members are taught in the art and can be
found, e.g., in Sulavik et al. (1995. Molecular Medicine. 1:436), Miller
and Sulavik (1996. Molecular Microbiology. 21:441) in which alignments of
MarR and related proteins are shown, or through the use of BLAST searches
and other techniques known in the art. Exemplary MarR family polypeptides
are also illustrated in the following chart:

[0031]Preferably, a MarR family polypeptide is MarR. Other preferred MarR
family polypeptides include: EmrR, Ec17kD, and MexR.

[0032]In a further embodiment, the MarR family polypeptide has a
winged-helix structure, such as the three dimensional structure of MarR.

[0033]FIG. 3 shows a sequence alignment of MarR with representative MarR
family polypeptides. The MarR secondary structure elements were
identified in its crystal structure and are illustrated in FIG. 3 (e.g.,
as tubes for α-helices (α) and arrows for β-sheets
(β) and the single wing region (W1)). The numbering in FIG. 3 is
according to the MarR primary sequence. Furthermore, residues that are
identical in all homologs are colored in red, highly conserved amino
acids are colored in yellow, and moderately conserved residues are
colored in blue. The MarR family polypeptides used for the alignment were
from the following organisms: MarR, E. coli; MprA (EmrR), E. coli; MexR,
Pseudomonas aeruginosa; YS87, Mycobacterium tuberculosis; SlyA,
Salmonella typhimurium; PecS, Erwinia chrysanthemi; CinR, Butyrivibrio
fibrisolvens. In a further embodiment, the MarR family polypeptide
comprises, consists essentially of, or consists of the polypeptide
sequence shown in Sequence Listing SEQ ID NO:1. Other MarR family
polypeptides of interest include EmrR, YS87, PecS, CinR, SlyA, Ec17kD,
MexR.

[0036]In one embodiment, the MarR family polypeptides of the invention are
naturally occurring. In another embodiment, the subject crystal
structures can be generated using non-naturally occurring forms of MarR
family polypeptides, e.g. mutants or synthetic forms of MarR family
polypeptides not found in nature.

[0037]In one embodiment, the MarR family polypeptide comprises one or more
conservative mutations as compared to the wild type protein for the
particular MarR family polypeptide. The term "MarR family polypeptide"
also includes fragments of MarR family polypeptides which minimally
retain at least a portion of the tertiary structure of the MarR family
protein.

[0038]MarR family member polypeptide sequences are "structurally related"
to one or more known MarR family members, preferably to MarR. This
structural relatedness is shown by sequence similarity between two MarR
family polypeptide sequences or between two MarR family nucleotide
sequences. Sequence similarity can be shown, e.g., by optimally aligning
MarR family member sequences using an alignment program for purposes of
comparison and comparing corresponding positions. To determine the degree
of similarity between sequences, they will be aligned for optimal
comparison purposes (e.g., gaps may be introduced in the sequence of one
protein or nucleic acid molecule for optimal alignment with the other
protein or nucleic acid molecules). The amino acid residues or bases and
corresponding amino acid positions or bases are then compared. When a
position in one sequence is occupied by the same amino acid residue or by
the same base as the corresponding position in the other sequence, then
the molecules are identical at that position. If amino acid residues are
not identical, they may be similar. An amino acid residue is "similar" to
another amino acid residue if the two amino acid residues are members of
the same family of residues having similar side chains. Families of amino
acid residues having similar side chains have been defined in the art
(see, for example, Altschul et al. 1990. J. Mol. Biol. 215:403) including
basic side chains (e.g., lysine, arginine, histidine), acidic side chains
(e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g.,
glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),
nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,
proline, phenylalanine, methionine, tryptophan), beta-branched side
chains (e.g., threonine, valine, isoleucine) and aromatic side chains
(e.g., tyrosine, phenylalanine, tryptophan). The degree (percentage) of
identity or similarity between sequences, therefore, can be calculated as
a function of the number of identical or similar positions shared by two
sequences (i.e., % homology=# of identical or similar positions/total #
of positions×100). Alignment strategies are well known in the art;
see, for example, Altschul et al. supra for optimal sequence alignment.

[0039]MarR family polypeptides share some amino acid sequence similarity
with MarR. The nucleic acid and amino acid sequences of MarR as well as
other MarR family polypeptides are available in the art. For example, the
nucleic acid and amino acid sequence of MarR can be found, e.g., on
GeneBank (accession number M96235 or in Cohen et al. 1993. J. Bacteriol.
175:1484, or in SEQ ID NO:1).

[0040]The nucleic acid and protein sequences of MarR can be used as "query
sequences" to perform a search against databases (e.g., either public or
private) to, for example, identify other MarR family members having
related sequences. Such searches can be performed, e.g., using the NBLAST
and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol.
Biol. 215:403-10. BLAST nucleotide searches can be performed with the
NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences
homologous to MarR family nucleic acid molecules. BLAST protein searches
can be performed with the XBLAST program, score=50, wordlength=3 to
obtain amino acid sequences homologous to MarR protein molecules of the
invention. To obtain gapped alignments for comparison purposes, Gapped
BLAST can be utilized as described in Altschul et al., (1997) Nucleic
Acids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST
programs, the default parameters of the respective programs (e.g., XBLAST
and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.

[0041]MarR family members can also be identified as being structurally
similar based on their ability to specifically hybridize to the
complement of nucleic acid sequences specifying MarR. Such stringent
conditions are known to those skilled in the art and can be found e.g.,
in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y.
(1989), 6.3.1-6.3.6. A preferred, non-limiting example of stringent
hybridization conditions are hybridization in 6× sodium
chloride/sodium citrate (SSC) at about 45° C., followed by one or
more washes in 0.2×SSC, 0.1% SDS at 50-65° C. Conditions for
hybridizations are largely dependent on the melting temperature that is
observed for half of the molecules of a substantially pure population of
a double-stranded nucleic acid. The melting temperature is the
temperature in ° C. at which half the molecules of a given
sequence are melted or single-stranded. For nucleic acids of sequence 11
to 23 bases, the melting temperature can be estimated in degrees C. as
2(number of A+T residues)+4(number of C+G residues). Hybridization or
annealing of nucleic acid molecules should be conducted at a temperature
lower than the melting temperature, e.g., 15° C., 20° C.,
25° C. or 30° C. lower than the melting temperature. The
effect of salt concentration (in M of NaCl) can also be calculated, see
for example, Brown, A., "Hybridization" pp. 503-506, in The Encyclopedia
of Molec. Biol., J. Kendrew, Ed., Blackwell, Oxford (1994).

[0042]Preferably, the nucleic acid sequence of a MarR family member
identified in this way is at least about 10%, 20%, more preferably at
least about 30%, more preferably at least about 40% identical and most
preferably at least about 50%, or 60% identical or more with a MarR
nucleotide sequence. Preferably, MarR family members have an amino acid
sequence at least about 20%, more preferably at least about 30%, more
preferably at least about 40% identical and most preferably at least
about 50%, or 60% or more identical with a MarR amino acid sequence.
However, it will be understood that the level of sequence similarity
among microbial regulators of gene transcription, even though members of
the same family, is not necessarily high. This is particularly true in
the case of divergent genomes where the level of sequence identity may be
low, e.g., less than 20% (e.g., B. burgdorferi as compared e.g., to B.
subtilis). For example, the level of amino acid sequence homology between
MarR and Pecs is about 31% and the level of amino acid sequence homology
between MarR and PapX is about 28% when determined as described above.
Accordingly, structural similarity among MarR family members can also be
determined based on "three-dimensional correspondence" of amino acid
residues.

[0043]The language "three-dimensional correspondence" includes residues
which spatially correspond, e.g., are in the same functional position of
a MarR family protein member as determined, e.g., by x-ray
crystallography, but which may not correspond when aligned using a linear
alignment program. The language "three-dimensional correspondence" also
includes residues which perform the same function, e.g., bind to DNA or
bind the same cofactor, as determined, e.g., by mutational analysis.
Thus, MarR family members can be identified based on functional homology
and sequence homology, e.g., as described in the art (Li et al. 2001.
EMBO Journal 20:4854).

[0044]Preferred MarR family polypeptides include: MarR, EmrR, Ec17kD,
MexR, PapX , SlyA, Hpr, PecS, Hpr, MprA, or (EmrR). In a more preferred
embodiment, a MarR family polypeptide is selected from the group
consisting of: MarR, EmrR, Ec17kD, and MexR. In a particularly preferred
embodiment, a MarR family polypeptide is MarR.

[0047]In addition to full length MarR family polypeptide fragments MarR
family polypeptide which are useful in making crystals are also within
the scope of the invention. Accordingly, MarR family polypeptides for use
in the instant screening assays can be full length MarR family member
proteins or fragments thereof. Thus, a MarR family polypeptide can
comprise, consist essentially of, or consist of an amino acid sequence
derived from the full length amino acid sequence of a MarR family member.
For example, in one embodiment, a polypeptide comprising a MarR family
polypeptide DNA interacting domain or a polypeptide comprising a MarR
family member protein interacting domain can be used.

[0048]In addition, naturally or non-naturally occurring variants of these
polypeptides and nucleic acid molecules which retain the same functional
activity, e.g., the ability to bind to DNA and regulate transcription.
Such variants can be made, e.g., by mutation using techniques which are
known in the art. Alternatively, variants can be chemically synthesized.

[0049]For example, it will be understood that the MarR family polypeptides
described herein, are also meant to include equivalents thereof. For
instance, mutant forms of MarR family polypeptides which are functionally
equivalent, (e.g., have the ability to bind to DNA and to regulate
transcription from an operon) can be made using techniques which are well
known in the art. Mutations can include, e.g., at least one of a discrete
point mutation which can give rise to a substitution, or by at least one
deletion or insertion. For example, random mutagenesis can be used.
Mutations can be made, e.g., by random mutagenesis or using cassette
mutagenesis. For the former, the entire coding region of a molecule is
mutagenized by one of several methods (chemical, PCR, doped
oligonucleotide synthesis) and that collection of randomly mutated
molecules is subjected to selection or screening procedures. In the
latter, discrete regions of a protein, corresponding either to defined
structural or functional determinants (e.g., the first or second helix of
a helix-turn-helix domain) are subjected to saturating or semi-random
mutagenesis and these mutagenized cassettes are re-introduced into the
context of the otherwise wild type allele. In one embodiment, PCR
mutagenesis can be used. For example, Megaprimer PCR can be used (O. H.
Landt, Gene 96:125-128).

[0050]In addition, other portions of the above described polypeptides
suitable for use in the claimed assays, such as those which retain their
function (e.g., the ability to bind to DNA, to regulate transcription
from an operon) or those which are critical for binding to regulatory
molecules (such as compounds) can be easily determined by one of ordinary
skill in the art (e.g, using standard truncation or mutagenesis
techniques) and used in the instant assays. Exemplary techniques are
described by Gallegos et al. (1996. J. Bacteriol. 178:6427).

[0051]It shall be understood that the instant invention also pertains to
isolated MarR family member polypeptides, portions thereof, and the
nucleic acid molecules encoding them, including naturally occurring and
mutant forms.

Preparation of MarR Family Polypeptides

[0052]Preferred MarR family polypeptides for use in screening assays are
synthesized, isolated or recombinant polypeptides. In one embodiment,
MarR family polypeptides can be made from nucleic acid molecules. Nucleic
acid molecules encoding MarR family polypeptides can be used to produce
MarR family polypeptides. For example, nucleic acid molecules encoding a
MarR family polypeptide can be isolated (e.g., isolated from the
sequences which naturally flank it in the genome and from cellular
components) and can be used to produce a MarR family polypeptide. In one
embodiment, a nucleic acid molecule which has been (1) amplified in vitro
by, for example, polymerase chain reaction (PCR); (2) recombinantly
produced by cloning, or (3) purified, as by cleavage and gel separation;
or (4) synthesized by, for example, chemical synthesis can be used to
produce MarR family polypeptides. The term "nucleic acid" refers to
polynucleotides such as deoxyribonucleic acid (DNA) and ribonucleic acid
(RNA).

[0053]Nucleic acid molecules specifying MarR family polypeptides can be
placed in a vector. The term "vector" refers to a nucleic acid molecule
capable of transporting another nucleic acid molecule to which it has
been linked. The term "expression vector" includes any vector, (e.g., a
plasmid, cosmid or phage chromosome) containing a gene construct in a
form suitable for expression by a cell (e.g., linked to a promoter). In
the present specification, "plasmid" and "vector" are used
interchangeably, as a plasmid is a commonly used form of vector.
Moreover, the invention is intended to include other vectors which serve
equivalent functions.

[0054]Exemplary expression vectors for expression of a gene encoding a
MarR family polypeptide and capable of replication in a bacterium, such a
bacterium from a genus selected from the group consisting of:
Escherichia, Bacillus, Streptomyces, Streptococcus, or in a cell of a
simple eukaryotic fungus such as a Saccharomyces or, Pichia, or in a cell
of a eukaryotic organism such as an insect, a bird, a mammal, or a plant,
are known in the art. Such vectors may carry functional
replication-specifying sequences (replicons) both for a host for
expression, for example a Streptomyces, and for a host, for example, E.
coli, for genetic manipulations and vector construction. See e.g. U.S.
Pat. No. 4,745,056. Suitable vectors for a variety of organisms are
described in Ausubel, F. et al., Short Protocols in Molecular Biology,
Wiley, New York (1995), and for example, for Pichia, can be obtained from
Invitrogen (Carlsbad, Calif.).

[0055]Useful expression control sequences, include, for example, the early
and late promoters of SV40, adenovirus or cytomegalovirus immediate early
promoter, the lac system, the trp system, the TAC or TRC system, T7
promoter whose expression is directed by T7 RNA polymerase, the major
operator and promoter regions of phage lambda, the control regions for fd
coat protein, the promoter for 3-phosphoglycerate kinase or other
glycolytic enzymes, the promoters of acid phosphatase, e.g., Pho5, the
promoters of the yeast α-mating factors, the polyhedron promoter of
the baculovirus system and other sequences known to control the
expression of genes of prokaryotic or eukaryotic cells or their viruses,
and various combinations thereof. A useful translational enhancer
sequence is described in U.S. Pat. No. 4,820,639.

[0056]It should be understood that the design of the expression vector may
depend on such factors as the choice of the host cell to be transformed
and/or the type of protein desired to be expressed.

[0057]"Transcriptional regulatory sequence" is a generic term to refer to
DNA sequences, such as initiation signals, enhancers, operators, and
promoters, which induce or control transcription of nucleic acid
sequences with which they are operably linked. It will also be understood
that a recombinant gene encoding a MarR family polypeptide can be under
the control of transcriptional regulatory sequences which are the same or
which are different from those sequences which control transcription of
the naturally-occurring MarR family gene. Exemplary regulatory sequences
are described in Goeddel; Gene Expression Technology Methods in
Enzymology 185, Academic Press, San Diego, Calif. (1990). For instance,
any of a wide variety of expression control sequences, that control the
expression of a DNA sequence when operatively linked to it, may be used
in these vectors to express DNA sequences encoding the MarR family
proteins of this invention.

[0058]Appropriate vectors are widely available commercially and it is
within the knowledge and discretion of one of ordinary skill in the art
to choose a vector which is appropriate for use with a given microbial
cell. The sequences encoding MarR family polypeptides can be introduced
into a cell on a self-replicating vector or may be introduced into the
chromosome of a microbe using homologous recombination or by an insertion
element such as a transposon.

[0059]Such vectors can be introduced into cells using standard techniques,
e.g., transformation or transfection. The terms "transformation" and
"transfection" mean the introduction of a nucleic acid, e.g., an
expression vector, into a recipient or "host" cell. The term
"transduction" means transfer of a nucleic acid sequence, preferably DNA,
from a donor to a recipient cell, by means of infection with a virus
previously grown in the donor, preferably a bacteriophage. Nucleic acids
can also be introduced into microbial cells by transformation using
calcium chloride or electroporation.

[0060]"Cells," "host cells," "recipient cells, are terms used
interchangeably herein. It is understood that such terms refer not only
to the particular subject cell but to the progeny or potential progeny of
such a cell. In preferred embodiments, cells used to express MarR family
polypeptides for purification, e.g., host cells, comprise a mutation
which renders any endogenous MarR family polypeptide nonfunctional or
causes the endogenous polypeptide to not be expressed. In other
embodiments, mutations may also be made in other related genes of the
host cell, such that there will be no interference from the endogenous
host loci.

[0061]Purification of a MarR family polypeptides, e.g., recombinantly
expressed polypeptides, can be accomplished using techniques known in the
art. For example, if the MarR family polypeptide is expressed in a form
that is secreted from cells, the medium can be collected. Alternatively,
if the MarR family polypeptide is expressed in a form that is retained by
cells, the host cells can be lysed to release the MarR family
polypeptide. Such spent medium or cell lysate can be used to concentrate
and purify the MarR family polypeptide. For example, the medium or lysate
can be passed over a column, e.g., a column to which antibodies specific
for the MarR family member polypeptide have been bound. Alternatively,
such antibodies can be specific for a non-MarR family member polypeptide
which has been fused to the MarR family polypeptide (e.g., as a tag) to
facilitate purification of the MarR family member polypeptide. Other
means of purifying MarR family member polypeptides are known in the art.

Architecture of the MarR-Salicylate Co-Crystal Structure

[0062]The term "three dimensional structure" includes both pictorial
representations of MarR family polypeptides (e.g., such as those shown
for MarR in the Figures) as well as atomic coordinates (e.g., such as
those given in FIG. 1 for MarR-salicylate cocrystal, or in FIG. 2 for
MarR) and other renditions of the shape, size, or symmetry of a MarR
family polypeptide of interest. In a further embodiment, the three
dimensional structure of the crystallized MarR family polypeptide is
determined to a resolution of 5 Å or better, 3 Å or better, 2.5
Å or better, or, advantageously, 2.3 Å or better. The three
dimensional structure of MarR, a MarR family polypeptide, is described in
greater detail below.

[0063]The salicylate containing MarR consists of a dimer with approximate
overall dimensions of 50×55×45 Å, as shown in FIG. 4.
There is one monomer in the asymmetric unit of the crystal with the dimer
composed of subunits related by a crystallographic two-fold rotation. The
dimeric structure is consistent with the results of earlier in vitro
experiments suggesting that MarR binds the mar operator (marO) as a dimer
(Martin, R. G. et al. supra (1996); Martin, R. G. & Rosner, J. L. Proc.
Natl. Acad. Sci. U.S.A. 92, 5456-5460 (1995)). Another family member,
MprA (EmrR) (FIG. 3) is also believed to function as a dimer (Brooun, A.,
et al. J. Bact. 181, 5131-5133 (1999)).

[0064]Each MarR salicylate subunit is an α/β protein with
approximate dimensions of 35×25×60 Å and can be divided
into two domains as shown in FIG. 4. FIG. 4 is a ribbon representation of
the co-crystal structure of the MarR dimer viewed with the subunit 2-fold
axis near vertical. The N- and C-terminal regions are closely juxtaposed
and intertwine with the equivalent regions of the second subunit to form
a domain that holds the subunits together (FIG. 5). This N-/C-terminal
domain is linked to the remainder of the protein by two long antiparallel
helices in each subunit. These helices lead to a globular domain that is
likely to be responsible for DNA binding (see below). Although the
globular DNA-binding domains of the dimer are adjacent to one another,
they make minimal contact with each other and are situated to function
independently. The overall organization of the N-/C-terminal domain and
the two DNA-binding domains results in the formation of an approximately
6 {acute over (Å)} wide channel through the center of the dimer
(FIGS. 6 and 7). The electrostatic surface potential is consistent with
the putative DNA-binding regions being strongly electropositive, as
observed in other such winged-helix DNA-binding proteins (Gajiwala, K. S.
& Burley, S. K. Curr. Opin. Str. Biol. 10, 110-116 (2000)).

[0065]Genetic and biochemical data have previously identified the
N-terminus of MarR to be important for mediating protein-protein contacts
between repressor subunits and have demonstrated that the C-terminus is
important for protein function (Alekshun, M. N., et al. Mol. Microbiol.
35, 1394-404 (2000); Linde, H. J. et al. supra). The present structure
shows that α-helices in the N- and C-terminal regions of each
monomer fold around and interdigitate with those of the other subunit to
form a well-packed hydrophobic core (FIG. 5) burying a surface area of
3,570 Å2 (the total buried surface area for the whole dimer is
3,700 Å2). The dimer is further stabilized in this region by
several intermolecular hydrogen bonds, notably that between the
ε-amino group of Lys 24 and the main chain carbonyl oxygen of Pro
144' in the C-terminus of the second subunit and that between the main
chain carbonyl oxygen of Glu 10 and the side chain amino group of Lys
140'.

[0066]While the DNA-binding lobe of each subunit also forms a well-packed
hydrophobic core, the only interactions between these lobes of the two
subunits are salt bridges formed between Asp 67 and Arg 73' and the
reciprocal pair (FIG. 8). These salt bridges stabilize the relationship
between the two lobes of the dimer in the crystal form of the protein but
if disrupted by other interactions, such as might occur during the
binding of MarR to marO, the two lobes would be able to act
independently. Relative movement of the lobes would require distortion of
the helices that link them to the N-/C-terminal domain. The long linker
helix region encompassing residues 103-126 (α5/α5') appears
poorly ordered in the region of Gly 116, as is the loop (residues
128-131) that connects this helix to the C-terminal helix
(α6/α6'). It is possible that flexibility at these sites in
MarR helps to accommodate relative shifts of the two lobes of the dimer
that might occur on binding to DNA.

Architecture of the MarR Crystal Structure

[0067]The MarR without salicylate structure is a dimer and both subunits
of the dimer are in the asymmetric unit. These individual subunits are
joined by protein-protein interactions mediated by amino acids within
both the N- and C-termini of the monomers. Like the MarR-salicylate
structure, MarR without salicylate is an α/β protein. The MarR
without salicylate structure is, however, conformationally different from
the salicylate bound protein in that the caliper created by the dimer is
more closed in the form of the protein without salicylate. Thus, the
channel through the center of the dimer has been lost.

[0068]The overall architecture of the MarR without salicylate structure is
comparable to that of the salicylate bound protein. The presumed DNA
binding lobes or domains are linked to the remainder of the protein by
two long α-helices. The positioning of the two DNA binding lobes in
the MarR without salicylate structure is fixed by hydrogen bonds between
the two lobes. This arrangement is believed to be mediated by
interactions between Asp 67 and Arg 77'. In addition, Asp 26 is involved
in hydrogen bonds with the side chains of Lys 44 and Lys 25. Together,
the presumed recognition helices within the DNA binds lobes overlap by
approximately one helical turn. FIG. 9 shows a ribbon representation of
the MarR dimer with the two-fold axis near vertical.

The DNA Binding Domain

[0069]Previous studies have shown the region spanning amino acids 61-121
in MarR to be required for its DNA binding activity (Alekshun, M. N et
al., supra, (2000)). In the crystal structure, amino acids 55-100
[β1-α3-α4-β2-W1 (wing)-β3] adopt the
winged-helix fold (Clark, K. L. et al. Nature 364, 412-420 (1993)). The
overall topology [H1 (α2)-S1 (β1)-H2 (α3)-H3 (α4,
recognition helix)-S2 (β2)-W1-S3 (β3)] of this region is
similar to other winged-helix DNA binding proteins (the terminology
applied for these and subsequent structural elements is according to
Gajiwala and Burley, supra (2000)) except that a third strand of sheet
present in most members of the group appears to be represented in this
MarR structure only by an interaction with Ile 55 (β1). The presence
of this residue as the third component in the sheet interaction is
similar to that observed in OmpR (Martinez-Hackert, E. & Stock, A. M.
Structure 5, 109-124 (1997)), a winged helix protein, where Leu 180
interacts with the two strands of the antiparallel sheet that forms part
of the "wing" in this transcription factor.

[0070]Within the winged-helix family of DNA-binding proteins, there are
multiple modes of DNA binding. Members such as HNF-3γ use the
recognition helix (H3) of the motif as the primary determinant for
DNA-protein interactions in the major groove, and a wing region(s) (W1)
to form minor groove or phosphodiester backbone nucleoprotein contacts
(Clark, K. L. et al. supra (1993)). Others, such as hRFX1, use W1 to
interact with the major groove and the H3 helix makes only a single minor
groove contact (Gajiwala, K. S. et al. Nature 403, 916-921 (2000)). The
juxtaposition of the DNA-binding lobes in the present structure does not
allow for modeling of the whole dimer onto a B-DNA representation of the
operator. However, since mutations in both α4 (H3) and W1 affect
the DNA binding activity of MarR it is expected that amino acids from
each of these regions would contribute to the DNA binding activity of the
protein. For example, mutations in α4, including an R73c change,
abolish MarR DNA binding activity in whole cells and in vitro (Alekshun,
M. N et al., supra, (2000)). In the present crystal structure, it is the
side chain of Arg 73 that is hydrogen bonded to Asp 67' of the other
subunit, an interaction that stabilizes the relative orientation of the
two DNA-binding lobes. Also, an R94C mutation at the tip of W1 is
inactive in a whole cell assay while a G95S "superrepressor" mutation
increases the DNA binding activity of MarR 30-fold in vitro (Alekshun, M.
N et al., supra, (2000); Alekshun, M. N. & Levy, S. B. J. Bact. 181,
3303-3306 (1999)). In the absence of protein-DNA co-crystal structures,
the precise mechanism by which these mutations affect the DNA binding
activity of the protein is uncertain.

[0071]Footprinting experiments have suggested that MarR binds as a dimer
at two separate but very similar sites in marO, the protein protects
˜21-bp of DNA on both strands at a single site, and does not bend
its target (Martin, R. G. et al., supra (1996); Martin, R. G. et al.
Proc. Natl. Acad. Sci. U.S.A. 92, 5456-5460 (1995)). Each MarR binding
site is composed of two half-sites whose organization is such that they
are on different faces of the DNA double helix (Alekshun, M. N. et al.
Mol. Microbiol. 35, 1394-404 (2000)), an arrangement that is very similar
to the hRFX1 binding site (Gajiwala, K. S. et al. Nature 403, 916-921
(2000)). For MarR to bind as a dimer, with each winged-helix DNA binding
domain contacting one half-site on B-DNA, geometric constraints suggest
only a few possible modes of binding. One scenario, involving the binding
of a single dimer to one MarR binding site, would require reorientation
of the DNA binding lobes so that each could reach one half-site. This
would be analogous to the binding of an E2F-DP heterodimer (a eukaryotic
transcription factor in which each subunit also has a winged-helix DNA
binding domain) to its cognate binding site (Zheng, N. et al. Genes Dev.
13, 666-74. (1999)). A second scenario would involve the binding of two
dimers, on opposite faces of the double helix, to a single MarR binding
site. This model would be analogous to the binding of DtxR (a bacterial
protein with a winged-helix DNA binding domain) to its target, although
in DtxR the half-sites are on the same face of the DNA helix (Pohl, E. et
al. J. Biol. Chem. 273, 22420-22427 (1998); White, A. et al. Nature 394,
502-506 (1998)).

[0072]The term "appropriate conditions" include those conditions which
result in the formation of a crystal which can by analyzed to a
resolution of 5.0 A or less. The crystals may be formed using suitable
art recognized techniques, such as hanging droplet vapor diffusion. In
one embodiment, the temperature of crystallization of the MarR family
polypeptide is from about 1° C. to about 30° C., from about
10° C. to about 25° C., from about 15° C. to about
20° C., or abut 17° C. In a further embodiment, the
conditions are selected such that crystals of said MarR family
polypeptide grow within an acceptable time and reach dimensions which are
suitable for structural determination, e.g., by using X-ray diffraction.
In one embodiment, the acceptable time is 8 weeks or less, 6 weeks or
less, 4 weeks or less, or 3 weeks or less. In an embodiment, the
dimensions of the crystal are approximately 0.1 mm or greater per side,
0.2 mm or greater per said, or approximately 0.3 mm per side or greater.

[0073]In a further embodiment, the appropriate conditions include a
cocrystallization agent which interacts with the protein such that the
three dimensional structure of the protein can be determined.

[0074]The term "cocrystallization agent" includes substances which can be
crystallized with the MarR family polypeptide such that the three
dimensional structure can be determined. In an embodiment, the
cocrystallization agent is a MarR family polypeptide modulator. The term
"MarR family polypeptide modulator" includes compounds which interact
with MarR family polypeptides, either to inhibit or enhance the activity
of the MarR family polypeptides, such that they alter its activity in its
non-crystallized form. In one embodiment, the MarR family polypeptide
modulator is a MarR inhibitor (e.g., salicylate, plombagin, or DNP). In
an embodiment, the concentration of the salicylate is about 100 mM or
less, 150 mM or less, 200 mM or less, or 250 mM or less.

[0075]The crystal structure or MarR has been solved using crystals grown
in the presence and in the absence of high concentrations (250 mM) of
sodium salicylate. This agent, at millimolar concentrations, is known to
inhibit MarR activity both in vitro and in whole cells (Alekshun, M. N.
supra (1999)). It is routinely used as a model inhibitor of MarR to
induce MarA expression in E. coli and S. typhimurium (Cohen, S. P. et al.
J. Bact. 175, 7856-7862 (1993); Sulavik, M. C. et al. J. Bact. 179,
1857-1866 (1997)) and thus, to confer a Mar phenotype (Alekshun, M. N.
supra (1999)). In one example, salicylate was included in the current
crystal growth conditions to provide stable crystals. In another example,
the crystal structure of MarR was determined using MarR without
salicylate.

[0076]Electron density that is consistent with bound salicylate is
apparent at two sites on each subunit in the present structure. These
sites are on the surface of the molecule on either side of the proposed
DNA-binding helix α4 (H3). In one site (SAL-A), the salicylate
hydroxyl is hydrogen bonded to the hydroxyl side chain of Thr 72 in the
α4 (H3) helix and the salicylate carboxylate hydrogen bonds to the
guanidinium group of Arg 86. In the other site (SAL-B), the salicylate
hydroxyl hydrogen bonds to the backbone carbonyl of Ala 70 and its
carboxyl hydrogen bonds to Arg 77. In each of these sites, the salicylate
ring sits over a hydrophobic side chain in the pocket; Pro 57 in SAL-A
and Met 74 in SAL-B and other surface hydrophobes are also located
laterally within 3.5 {acute over (Å)} of the unsubstituted side of
the ring. Although SAL-B is solvent exposed, SAL-A packs in the crystal
with Val 96 of a symmetry mate situated 3.6 Å above the salicylate
ring and adjacent to the SAL-A site of this symmetry mate. Since both
SAL-A and SAL-B are close to the DNA binding helix, they may be
positioned to influence DNA binding.

[0077]The crystal structure of MarR was solved by multiwavelength
anomalous dispersion methods using protein containing selenomethionine.
Diffraction data were collected to 2.3 Å from crystals of both seleno
and native protein.

Use of the MarR Crystal Structure to Model the Structures of Other MarR
Family Polypeptides

[0078]In one embodiment, the invention pertains to a method for
determining the structure of a MarR family polypeptide comprising
analyzing the sequence of the related polypeptide and then modeling its
structure based on the structure of MarR. The invention also pertains to
the use of the MarR family polypeptide structures in the methods
described below, e.g., for the identification of MarR family polypeptide
modulating compounds.

[0079]Given the sequence-structure relationship described, the MarR
crystal structure (described below) in the presence or absence of a MarR
family polypeptide modulating compound can be used as a template to
generate a computational three-dimensional model of any of the other
members of the MarR protein family. In another embodiment, both crystals
can be compared and the resulting information (including information
regarding the binding site of the MarR family modulator) can be used. The
resulting structure(s) can be subjected to the entire complement of
computational approaches discussed and demonstrated above. Computer
software packages such as COMPOSER (SYBYL. Tripos, Inc. 1699 Hanley Rd.
St. Louis, Mo. 63144; Sutcliffe, M. J. et al. Protein Eng. 1987, 1,
385-392), MODELLER (Accelrys, Inc. 9685 Scranton Road San Diego, Calif.
92121-3752 U.S.A.; Sali, A. B. J. Mol. Biol. 1993, 234, 779-815) are
widely utilized. The process of generating a structure as described is
known as homology modeling or comparative molecular modeling.
Generically, the process includes overall protein sequence alignment,
determination of structurally conserved regions (SCR's), transposition of
the template structure onto the undetermined sequence, loop building and
refinement. As an example of how the MarR structure can be used for this
purpose, a three-dimensional model of SlyA was generated as described in
the appended examples.

[0080]The term "MarR family modulating compound" includes small molecules
and other chemical entities which are capable of modulating, e.g.,
increasing or decreasing or otherwise altering the activity of a MarR
family polypeptide or its down stream products, e.g., a MarR modulating
compound may modulate the binding of MarR to DNA (e.g., the marO operon)
or otherwise alter the expression of MarA. In one embodiment, the MarR
family modulating compound is a MarR activator that enhances the binding
of MarR to DNA (e.g., the marO operon), such that MarA expression is
reduced.

[0081]The term "MarR family modulating compound candidate" includes
compounds which are being screened or otherwise tested (e.g.,
computationally or in the laboratory) to determine whether or not they
modulate MarR or a MarR family polypeptide.

[0082]The term "rational drug design" includes both computer aided and
non-computer techniques where a protein is analyzed for active sites, and
then modulating compound candidates are designed to interact with the
particular spatial and electrochemical requirements of the particular
site.

[0083]The term "active site" includes regions of a protein where a MarR
family modulating compound physically interacts with a MarR family
polypeptide. Any portion of the surface of a MarR family polypeptide can
be considered an active site region or locus. In one embodiment, the
portion of the MarR family protein immediately adjacent to the binding
site of a MarR family modulating compound (e.g., a salicylate moiety) is
referred to as the active site for the MarR family polypeptide. Other
active sites include the DNA binding regions and regions necessary for
interactions with other biological components, e.g., DNA or protein.

[0084]The term "interacts" includes interactions between the MarR family
polypeptide and the MarR family modulating compound which result in
modulation of a MarR family associated activity, e.g., expression of MarA
when the MarR family polypeptide is MarR. The term also includes
interactions which are determined by the shape and electronic
complementarity between the MarR family polypeptide and the MarR family
modulating compound. The term "interact" includes detectable interactions
between molecules. The term interact is also meant to include "binding"
interactions between molecules. Exemplary interactions include
protein-protein and protein-nucleic acid interactions.

[0086]In these approaches, the active site is postulated and then placed
within a three-dimensional lattice of evenly distributed grid points. A
small molecular fragment or atom is placed on each lattice point, and a
mathematical evaluation is made to determine the electronic and spatial
properties at that point. After each lattice point within the active site
is thus defined, the spatial and electronic "values" are contoured to
generate maps or graphical representations that indicate the locations
within the active site that are capable of accommodating additional
"atomic bulk" and whether the atomic bulk should be charge positive,
negative or neutral. It is the general theory that "filling" the active
site with appropriate "atomic bulk" will optimize the drug-target
interaction, thereby producing the maximal pharmacological response.

[0087]For example, the program LigBuilder was used to characterize one of
the MarR active sites (SAL-A) in terms of its spatial and electronic
properties. The results from this program represent a collection of
colored crosses that depict an "inverse cast" of the MarR active site.
Each cross represents a point where a mathematical determination was
made. The shape of the inverse cast is dependent upon the van der Waals
radii of the target's atoms constituting the active site as defined by
the crystal structure of MarR. The colors indicate where the active site
prefers positive or negative charge complemetarity. For example, arginine
#86 of MarR is positively charged at physiologic pH. Consequently, atoms
or atom fragments that are negatively charged would produce the optimal
complimentarily about that point, which is correctly depicted by the
LigBuilder program.

[0088]Once the active site has been graphically defined, the spatial and
electronic representations of a MarR family modulating compound candidate
can be fit or docked within the target active site. Specific
modifications of an initial candidate can be made electronically, and
then tested to determine whether the complementarity between the active
site and the modulating compound candidate has been increased.

[0089]To demonstrate the use of the crystal structure for docking, the
coordinates of the salicylate (a MarR modulator, which, in one
embodiment, can be cocrystallized with MarR) were artificially removed
from the MarR active site. Using this newly created empty active site as
input, the program FLEXX is able to predict the proper binding
orientation of salicylate with MarR (FLEXX Module, in SYBYL. Tripos, Inc.
1699 Hanley Rd. St. Louis, Mo. 63144. Rarey, M. et al. J. Mol. Bio. 1996,
261, 470-489). The result of this docking experiment is shown in green,
which can be compared to the original salicylate orientation as
determined crystallographically. As shown, the dominant molecular
interactions between the cocrystallized salicylate molecule and the
active site residues are predicted by the docking algorithm, e.g. the
carboxylate and hydroxyl groups. The greatest variation between the
computationally predicted docking and that determined by experiment
occurs at the 4- and 5-positions of the aromatic ring. These correspond
to the regions of the cocrystallized salicylate molecule with the largest
crystallographic b-factors, and indicate that the carboxylate and
hydroxyl groups of the salicylate moiety create the primary interactions
within the active site. These two major interactions create a hinge point
where the aromatic ring pivots within the active site.

[0090]As used in a drug discovery program, small modifications of the
salicylate molecule can be made computationally, and then subjected to
the identical FLEXX docking as demonstrated above for salicylate. The
score of the modified salicylate can be compared to the original to
ascertain the modification's benefit to overall target site
complimentarily. This is a time consuming process, and is typically
utilized only as part of the lead optimization process in an active drug
discovery program. A variant of this approach is the automated
application to large virtual libraries of potential drug candidates,
known as automated ligand docking (Muegge, I.; Rarey, M. Small Molecule
Docking and Scoring. Reviews in Computational Chemistry; Wiley: New York,
2000). This approach is typically employed as a part of the lead
identification or screening process of a drug discovery program, since a
large number of modulating compound candidates can quickly be assessed
for active site complimentarily. Programs available include, but are not
restricted to DOCK (DOCK Suite of Programs: Reagents of the University of
California: DesJarlais et al. J. Med. Chem. 1988, 31, 722-729), AUTO_DOCK
(AUTO_DOCK: Olson, A. J., SCRIPPS, La Jolla, Calif. Goodsell, D. S., et
al. J. Mol. Recognit. 1996, 9), GLIDE, and FLEXX (FLEXX Module, SYBYL.
Tripos, Inc.; Rarey, M. et al. J. Mol. Bio. 1996, 261, 470-489). Each
requires an electronic representation of a library of potential drug
candidates. Early versions of these approaches treated the drug candidate
as a rigid body, wherein conformational flexibility was neglected.
Algorithmic improvements and increases in computational speed now allow
the flexibility of a potential candidate to be included. Based on the
relative values of these scores, a virtual library of structures can be
quickly screened for members that would produce the best interaction
within the active site. As such, large libraries, originating from
commercial vendors, from proprietary template enumeration or other
sources, can be culled to eliminate compounds that are not promising
(data reduction) and/or prioritized to highlight compounds warranting
further consideration. Compounds with better overall docking scores will
be placed higher on the list.

[0091]Each of the techniques described above are included as rational drug
design methods. Other rational drug design techniques include de novo
drug design which utilizes the structure of the protein to generate
molecules to dock within the active site. In this approach, a "seed"
atom, or seed-molecule with pre-defined attachment points is placed
within the active site. Programs are available to systematically "grow"
chemical modifications at the attachment points resulting in novel
molecules. Through an iterative process of growing and assessing the
complimentarily of the new structures, productive attachments can be
saved, while unproductive attachments are discarded. Subsequent
redefinition of the seed based on productive attachments can produce
large number of drug candidates for the specified target. This is an
unbiased approach since the resulting compound is not taken from a
pre-existing virtual library, and is often used to generate compounds
that would otherwise not be considered based on current proprietary
knowledge or chemist's intuition. For example, this approach was applied
to one of the MarR active sites using the program LigBuilder to produce a
list of novel potential drug candidates. The compounds generated by
LigBuilder are merely representative of one class of compounds which may
be useful as MarR family protein modulating compounds. The invention also
pertains to other compounds which may interact with other portions and
thus have little or no structural similarity to these compounds.

[0092]Rational drug design also may involve the identification of
pharmacophoric elements. In drug design, important functional groups are
referred to as pharmacophoric elements and are useful for productive
drug-target interaction. For example, for MarR salicylate site A (SAL-A),
certain interactions between the salicylate moiety and the MarR active
site may be attributable to the two main functional groups of the
salicylate moiety, namely the carboxylate and the hydroxyl groups. At
this site, the carboxylate creates a charge-charge interaction with
arginine #86, and the hydroxyl group interacts strongly with threonine
#72 by virtue of hydrogen bonding. Furthermore, the absence of either of
these elements may diminish the degree of complementarity. The collection
of pharmacophoric elements and their mutual spatial disposition within
the active site defines the pharmacophore of the active site (See, e.g.,
WO 97/27219). In one embodiment, a MarR family modulating compound of the
invention interacts with an amino acid corresponding (e.g., linearly or
three dimensionally) to arginine at position #86 of SEQ ID NO:1 and/or
threonine at position #72 SEQ ID NO:1.

[0093]For MarR, the carboxylate and hydroxyl groups of an inhibitor are
separated by a distance of about 1.5 Å. As such, any compound with a
similar functional groups thus positioned will possess the pharmacophore
for MarR. Such information can be deduced from a known collection of
compounds that demonstrate interaction with MarR. However, the crystal
structure of MarR and its active site can be used to define a series of
testable pharmacophore hypothesis. Programs, such as CoMFA, GRID and
LigBuilder are instrumental in defining these hypotheses in a manner
similar to that detailed by Clackson. In one embodiment, a MarR family
modulating compound of the invention comprises a carboxylate and a
hydroxyl group separated by a distance of 1.5 Angstroms.

[0094]In one embodiment, a known drug candidate is co-crystallized in the
active site (e.g., salicylate, plumbagin, or DNP for MarR), since the
exact coordinates of the pharmacophore can be determined. In another
embodiment, the MarR family member is crystallized without a
cocrystallizing agent. In another embodiment, the crystal structures of
the MarR family member in the presence and absence of the
co-crystallizing agent are compared to determine the effect of binding of
the cocrystallizing agent. Thus, with or without a co-crystallizing
compound, the pharmacophore can be used as a search query to identify
structures from virtual libraries of commercial (known) or hypothetical
structures. Programs including, but not restricted to UNITY (UNITY
Module, in SYBYL. Tripos, Inc. 1699 Hanley Rd. St. Louis, Mo. 63144),
CATALYST (CATALYST, Accelrys, Inc. 9685 Scranton Road San Diego, Calif.
92121-3752 U.S.A. Sprague, P. W. Comput.-Assisted Lead Find. Optim.,
[Eur. Symp. Quant. Struct.-Act. Relat.] 1997, 225-240) may be used for
this purpose (Greer, J. et al. J. Med. Chem. 1994, 37, 1035-1054; WO
99/45389). The pharmacophore elements can also be used as the seeds for
de novo design. LigBuilder was applied to the active site of MarR using
the carboxylate and hydroxyl groups as "seed" groups to approximate the
pharmacophore hypothesis. Common among these structures are the actual
elements of the pharmacophore as expected, but in nearly all of the
structures examined, another hydrogen bond acceptor was present,
indicating the possibility of yet another pharmacophoric element in the
pharmacophore.

[0095]In another embodiment, the invention pertains to a method for
identifying a MarR family modulating compound using the three-dimensional
structure of a MarR family polypeptide. The method includes selecting a
candidate MarR family modulating compound by performing rational drug
design with the set of atomic coordinates in FIG. 1 and/or FIG. 2 using
computer aided techniques, as described herein. In one embodiment, the
method also includes contacting the candidate MarR family modulating
compound with a MarR family peptide, and a nucleic acid molecule, and
then measuring the binding affinity of the MarR family polypeptide
peptide with the nucleic acid molecule, such that MarR family modulating
compounds are identified. In one embodiment, the nucleic acid molecule is
a nucleic acid molecule to which a particular MarR family member is known
to bind. For example, for MarR, the nucleic acid used for the binding
acid may be, for example, marO.

[0096]In a further embodiment, the MarR family modulating compound is a
MarR activator that acts, e.g., to inhibit the expression of MarA.

[0097]The invention also pertains to a method of identifying a MarR family
member modulating compound. The method includes obtaining a set of atomic
coordinates defining the three-dimensional structure of MarR or a MarR
family polypeptide; selecting a candidate MarR family modulating compound
by performing rational drug design with said three dimensional structure
of the MarR family polypeptide; contacting said candidate MarR family
modulating compound with MarR family polypeptide; and measuring the
ability of the candidate MarR family modulating compound to modulate the
activity of the MarR family polypeptide, thus identifying a MarR
modulating compound.

[0098]In one embodiment, the rational drug design is aided by a computer
program described supra. In one embodiment, the MarR family polypeptide
is MarR and has the polypeptide sequence given in SEQ ID NO. 1 and has
the atomic coordinates given in FIG. 1, when cocrystallized with
salicylate or FIG. 2, when crystallized without.

[0099]In another embodiment, the invention pertains to compounds generated
by the methods of the invention, described above. For example, the
invention pertains to the MarR family modulating compounds and MarR
modulating compounds generated by the rational drug design techniques
described above. Examples of MarR modulating compounds include those of
the formula (I):

X--Y--Z (I)

[0100]wherein [0101]X is an interacting moiety; [0102]Y is a hydrophobic
moiety; and [0103]Z is a polar moiety.

[0104]The term "interacting moiety" includes moieties which are capable of
interacting with a MarR family member. Preferably, such interacting
moieties interact with Thr 72 of SEQ ID. 1 or an amino acid molecule that
corresponds to Thr 72 in a MarR family polypeptide. In a further
embodiment, the interacting moiety is capable of interacting by hydrogen
bonding. Examples of interacting moieties include, but are not limited
to, hydroxyl, thiol, sulfanyl, sulfonyl, amino, carbonyl, alkyl, and acyl
moieties. The term "interacting moiety" includes moieties which allow the
MarR modulating compound to perform its intended function, e.g., modulate
MarR family member activity. In a further embodiment, the interacting
moiety is hydroxy, thiol, or amino.

[0105]The term "hydrophobic moiety" includes moieties which are capable of
interacting with the MarR family polypeptide such that the compound is
capable of performing its intended function, e.g., modulate MarR. In
certain embodiments, the hydrophobic moiety may be substituted with
substituents capable of hydrogen bonding such as, but not limited to,
hydroxy, thiol, carbonyl, amino, carboxylate, or thiol. Examples of
hydrophobic moieties include, but are not limited to, substituted and
unsubstituted alkyl, alkenyl, alkynyl, and aryl moieties.

[0106]In certain embodiments, the hydrophobic moiety is aryl. The aryl
moiety may be cyclic, bicyclic or tricyclic. Preferably, the hydrophobic
moiety is selected such that it is capable of interacting with MarR, such
that its activity is modulated. In a further embodiment, the MarR family
modulating compound is selected such that it is capable of interacting
with hydrophobic or neutral amino acid residues, such as, but not limited
to, Pro 57 or Met 74 or an amino acid residue corresponding to these
amino acids of SEQ NO:1.

[0107]The term "polar moiety" includes moieties which are capable of
interacting with MarR family polypeptide such that the activity of the
MarR family polypeptide is modulated. In one embodiment, the polar moiety
interacts with Arg 86 or Arg 77 or an amino acid residue corresponding to
these amino acids of SEQ ID NO:1. In one embodiment, polar moiety is
negatively charged. Examples of polar moieties include carboxylate and
isoteres thereof. Other examples include, but are not limited to,
phosphate, phosphite, sulfate, sulfite, nitrate, nitrite, nitro, hydroxy,
oxalate, and perchlororate.

[0108]In one embodiment, the MarR family modulating compound is a MarR
inhibitor. In another embodiment, the polar moiety and the interacting
moiety are separated by a distance of about 1.5 Angstroms.

[0109]In a further embodiment, the MarR modulating compound is of the
formula:

##STR00001##

wherein Y is a substituted or unsubstituted cyclic or bicyclic moiety, and
pharmaceutically acceptable salts and esters thereof. In a further
embodiment, X is hydroxyl. In another further embodiment, Y is monocyclic
or bicyclic, optionally substituted with a hydrophilic substituent.
Examples of MarR modulating compounds include those listed below.

##STR00002## ##STR00003## ##STR00004##

[0110]The compounds described herein can be synthesized by methods known
in the art. An ordinarily skilled artisan will be able to consult the
chemical literature and will be able to synthesize the compounds
described herein.

[0111]The term "alkenyl" includes unsaturated aliphatic groups, including
straight-chain alkenyl groups, branched-chain alkenyl groups,
cycloalkenyl (alicyclic) groups, alkenyl substituted cycloalkyl or
cycloalkenyl groups, and cycloalkenyl substituted alkyl or alkenyl
groups. The term alkenyl further includes alkenyl groups, which can
further include oxygen, nitrogen, sulfur or phosphorous atoms replacing
one or more carbons of the hydrocarbon backbone, e.g., oxygen, nitrogen,
sulfur or phosphorous atoms. In preferred embodiments, a straight chain
or branched chain alkenyl group has 10 or fewer carbon atoms in its
backbone (e.g., C1-C10 for straight chain, C3-C10 for
branched chain), and more preferably 6 or fewer. Likewise, preferred
cycloalkenyl groups have from 4-7 carbon atoms in their ring structure,
and more preferably have 5 or 6 carbons in the ring structure, e.g.,
cyclopentene or cyclohexene.

[0112]The term "alkyl" includes saturated aliphatic groups, including
straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl
(alicyclic) groups, alkyl substituted cycloalkyl groups, and cycloalkyl
substituted alkyl groups. The term alkyl further includes alkyl groups,
which can further include oxygen, nitrogen, sulfur or phosphorous atoms
replacing one or more carbons of the hydrocarbon backbone, e.g., oxygen,
nitrogen, sulfur or phosphorous atoms. In preferred embodiments, a
straight chain or branched chain alkyl has 10 or fewer carbon atoms in
its backbone (e.g., C1-C10 for straight chain, C3-C10
for branched chain), and more preferably 6 or fewer.

[0113]Likewise, preferred cycloalkyls have from 4-7 carbon atoms in their
ring structure, and more preferably have 5 or 6 carbons in the ring
structure.

[0114]Moreover, the term alkyl includes both "unsubstituted alkyls" and
"substituted alkyls", the latter of which refers to alkyl moieties having
substituents replacing a hydrogen on one or more carbons of the
hydrocarbon backbone. Such substituents can include, for example,
halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,
aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl,
aminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato,
phosphinato, cyano, amino (including alkyl amino, dialkylamino,
arylamino, diarylamino, and alkylarylamino), acylamino (including
alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino,
imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates,
sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,
heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety. It will
be understood by those skilled in the art that the moieties substituted
on the hydrocarbon chain can themselves be substituted, if appropriate.
Cycloalkyls can be further substituted, e.g., with the substituents
described above. An "alkylaryl" moiety is an alkyl substituted with an
aryl (e.g., phenylmethyl (benzyl)).

[0115]The term "aryl" includes aryl groups, including 5- and 6-membered
single-ring aromatic groups that may include from zero to four
heteroatoms, for example, benzene, pyrrole, furan, thiophene, imidazole,
benzoxazole, benzothiazole, triazole, tetrazole, pyrazole, pyridine,
pyrazine, pyridazine and pyrimidine, and the like. Aryl groups also
include polycyclic fused aromatic groups such as naphthyl, quinolyl,
indolyl, and the like. Those aryl groups having heteroatoms in the ring
structure may also be referred to as "aryl heterocycles", "heteroaryls"
or "heteroaromatics". The aromatic ring can be substituted at one or more
ring positions with such substituents as described above, as for example,
halogen, hydroxyl, alkoxy, alkylcarbonyloxy, arylcarbonyloxy,
alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,
alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, phosphate, phosphonato,
phosphinato, cyano, amino (including alkyl amino, dialkylamino,
arylamino, diarylamino, and alkylarylamino), acylamino (including
alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino,
imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates,
sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,
heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety. Aryl
groups can also be fused or bridged with alicyclic or heterocyclic rings
which are not aromatic so as to form a polycycle (e.g., tetralin).

[0116]The terms "alkenyl" and "alkynyl" include unsaturated aliphatic
groups analogous in length and possible substitution to the alkyls
described above, but that contain at least one double or triple bond,
respectively. Examples of substituents of alkynyl groups include, for
example alkyl, alkenyl (e.g., cycloalkenyl, e.g., cyclohenxenyl), and
aryl groups.

[0117]Unless the number of carbons is otherwise specified, "lower alkyl"
includes an alkyl group, as defined above, but having from one to three
carbon atoms in its backbone structure. Likewise, "lower alkenyl" and
"lower alkynyl" have similar chain lengths.

[0118]The terms "alkoxyalkyl", "polyaminoalkyl" and "thioalkoxyalkyl"
include alkyl groups, as described above, which further include oxygen,
nitrogen or sulfur atoms replacing one or more carbons of the hydrocarbon
backbone, e.g., oxygen, nitrogen or sulfur atoms.

[0119]The terms "polycyclyl" or "polycyclic radical" refer to two or more
cyclic rings (e.g., cycloalkyls, cycloalkenyls, aryls and/or
heterocyclyls) in which two or more carbons are common to two adjoining
rings, e.g., the rings are "fused rings". Rings that are joined through
non-adjacent atoms are termed "bridged" rings. Each of the rings of the
polycycle can be substituted with such substituents as described above,
as for example, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,
alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,
alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,
phosphonato, phosphinato, cyano, amino (including alkyl amino,
dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino
(including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),
amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,
sulfates, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl,
cyano, azido, heterocyclyl, alkyl, alkylaryl, or an aromatic or
heteroaromatic moiety.

[0120]The term "heteroatom" includes atoms of any element other than
carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, sulfur
and phosphorus.

[0121]The term "alkylsulfinyl" include groups which have one or more
sulfinyl (SO) linkages, typically 1 to about 5 or 6 sulfinyl linkages.
Advantageous alkylsulfinyl groups include groups having 1 to about 12
carbon atoms, preferably from 1 to about 6 carbon atoms.

[0122]The term "alkylsulfonyl" includes groups which have one or more
sulfonyl (SO2) linkages, typically 1 to about 5 or 6 sulfonyl
linkages. Advantageous alkylsulfonyl groups include groups having 1 to
about 12 carbon atoms, preferably from 1 to about 6 carbon atoms.

[0123]The term "alkanoyl" includes groups having 1 to about 4 or 5
carbonyl groups. The term "aroyl" includes aryl groups, such as phenyl
and other carbocyclic aryls, which have carbonyl substituents. The term
"alkaroyl" includes aryl groups with alkylcarbonyl substituents, e.g.,
phenylacetyl.

[0124]The invention also includes a method for inhibiting expression of
MarA. The method includes contacting MarR with a MarR inhibiting
compound. In an embodiment, the MarR inhibiting compound is of the
formula (I):

X--Y--Z (I)

wherein X is an interacting moiety, Y is a hydrophobic moiety; and Z is a
polar moiety, and acceptable salts thereof. In an embodiment, the MarR
inhibiting compound inhibits the binding of MarR to DNA (e.g., the marO
operon).

[0125]Biological systems generally function through carefully
choreographed interactions of their respective components. The operative
mechanisms for many disease states implicate protein-protein interactions
as key. For transcription factors, such as MarR, protein-DNA and
protein-RNA interactions control the regulation events for the biological
system. The drug design approaches discussed above are targeted in part
to disrupt the interaction between MarR and the mar operon. Knowledge of
the three dimensional structure of the MarR-marO complex can provide
clues as to the key interactions (pharmacophore) made between them. A
computer model of an interaction between MarR and DNA is shown in FIG.
10.

[0126]The invention also pertains to a method for decreasing multidrug
resistance in a microbe, e.g., E. coli. The invention includes contacting
E. coli with a MarR inhibiting compound, such that said multidrug
resistance in E. coli is decreased. In an embodiment, the MarR inhibiting
compound is of the formula (I):

X--Y--Z (I)

wherein X is an interacting moiety; Y is a hydrophobic moiety; and Z is a
polar moiety, and acceptable salts thereof.

[0127]The invention also pertains to methods for modulating activity of a
MarR family polypeptide. The method includes contacting a MarR family
polypeptide with a MarR family modulating compound identified by any
method described herein (e.g., the computer modeling techniques, etc.).
The invention also pertains to any compound discovered using techniques
described herein.

[0128]The invention is further illustrated by the following examples,
which should not be construed as further limiting. The contents of all
references, pending patent applications and published patents, cited
throughout this application are hereby expressly incorporated by
reference.

[0135]Diffraction data were collected at the Brookhaven National.
Synchrotron Light Source, beamline X8C. Crystals were flash frozen in
mother liquor at the beam line before data collection. All data were
processed and reduced using DENZO and SCALEPACK (Otwinowski, Z. In CCP4
Proceedings. 56-62 (Daresbury Laboratory, Warrington, UK, 1993). The
space group of the MarR-salicylate co-crystals was determined to be
I4122 with one molecule in the asymmetric unit and with unit cell
dimensions of a=b=62.0 Å, c=132.9 Å,
α=β=γ=90° for both the native and the
selenoprotein. Data were collected on the selenoprotein crystals at three
wavelengths to enable MAD phasing. Phases were determined from the MAD
data using the program SOLVE (Terwilliger, T. C. & Berendzen, J. Acta
Crystallogr. D. 55, 849-861 (1999)). This showed two selenium sites per
asymmetric unit, with the third selenomethionine, at the N-terminus,
apparently disordered. Maps were solvent-flattened using the program DM
and the model was built into density using the program O (Collaborative
Computational Project, Number 4. Acta Crystallogr. D. 50, 760-763 (1994);
Jones, T. A. et al. Acta Crystallogr. A 47, 110-119 (1991)). Model and
refinement parameters for salicylate were obtained from the Hetero
Compound Information Center (Kleywegt, G. J. & Jones, T. A. Acta
Crystallogr. D. 54, 1119-1131 (1998)). Model refinement was performed
using CNS and cycles of rebuilding and refinement continued to give the
final model (Brunger, A. T. et al. Acta Crystallogr. D. 54, 905-921
(1998)). Model quality was assessed by sa-omit, Fo-Fc, maps generated
over the whole molecule omitting no more than 7% of the structure at a
time. The model extends from residue 6 to the C-terminus at residue 144.
In common with several other transcription factors (e.g. TetR, (1A6I),
ArgR (1B4B) and TreR (1BYK)), MarR shows relatively high thermal mobility
throughout the structure, as reflected by the B-factors. Certain regions
appear to be particularly mobile, including the extended structure at the
N-terminus, the tip of the "wing" (residues 91-94), parts of the α5
helix, especially around Gly 116 and the connecting loop (128-131)
between the α5 and the C-terminal α6 helix. Consistent with
the high B-factors, the molecule shows few well-ordered solvent
molecules. PROCHECK reports overall g-factors of 0.25 (dihedrals) and
0.55 (main chain covalent forces) and shows that 91% of the residues fall
within the most favored region of the Ramachandran plot, with only
residue Ala 53 in a disallowed region. This residue is located at the
start of the loop connecting the α2 and α3 helices.

[0136]The coordinates of the MarR-salicylate cocrystal are shown in FIG.
1. Data collection, phasing and refinement statistics for the MarR-sal
cocrystal structure is shown in Table 1.

[0141]Diffraction data were collected at the Brookhaven National
Synchrotron Light Source, beamline X8C. Crystals were flash frozen in
mother liquor at the beam line before data collection. All data were
processed and reduced using DENZO and SCALEPACK (Otwinowski, Z. In CCP4
Proceedings. 56-62 (Daresbury Laboratory, Warrington, UK, 1993).

[0142]The coordinates of the MarR crystal without salicylate are shown in
FIG. 2. Data collection, phasing, and refinement statistics for the MarR
co-crystal structure is shown in Table 2.

Use of the Crystal Structure of MarR to Model Other MarR Family
Polypeptides

[0143]The amino acid sequences of MarR and SlyA are shown in FIG. 11. This
alignment is generated automatically using the subroutines in COMPOSER,
however it can be generated by a variety of other programs. FIG. 12 shows
the results of the COMPOSER program in identifying the structurally
conserved regions (SCRs).

[0144]The amino acids colored magenta are the regions of MarR and SlyA
where the amino acid sequences are predicted to exhibit the same tertiary
structure. These predictions are based on a knowledge base of information
derived from the compilation of known crystal structures. Specifically,
statistical correlations are made for protein tertiary structure with the
respective amino acid sequences, and it was found that the correlations
could be used in a predictive manner.

[0145]In the comparative molecular modeling process, the three-dimensional
coordinates of the MarR backbone in the SCRs were directly transposed to
create a general framework for SlyA as seen in FIGS. 13a and 13b. FIG.
13a is the Ca-trace of MarR with the SCRs highlighted as orange
tubes. The SCRs were "extracted" in their same mutual orientation to
produce the basic framework of SlyA, which is shown in FIG. 13B. The
process at this point generally includes only the backbone chain
coordinates; the sidechains are added computationally to the SCR's on the
left to create the SlyA protein. This model can, in all respects, be
subjected to the identical regimen of computational protocols as the bona
fide MarR crystal structure (Podlogar, B. L. et al. J. Med. Chem. 1997,
40, 3453-3455).

[0146]The regions in yellow (FIG. 12) are the "loops" that connect the
SCRs. Loop regions, in general, exhibit the greatest variation among
members in the same family. As such, no logical template for their
construction is available. Again, use is made of the vast knowledge
contained in the database of determined protein structures to construct
the loop regions. FIG. 14 shows the fully constructed SlyA structure
(purple) in comparison to the template protein, MarR.

Example 4

Use of the Computer Modeling to Characterize the MarR Active Site

[0147]For example, the program LigBuilder was used to characterize the
MarR active site in terms of its spatial and electronic properties. The
results represent a collection of colored crosses that depict an "inverse
cast" of the MarR active site. Each cross represents a point where a
mathematical determination was made. The shape of the inverse cast is
dependent upon the van der Waals radii of the target's atoms constituting
the active site as defined by the crystal structure of MarR. The colors
indicate where the active site prefers positive or negative charge
complemetarity. For example, arginine #86 of MarR is positively charged
at physiologic pH. Consequently, atoms or atom fragments that are
negatively charged would produce the optimal complimentarily about that
point, which is correctly depicted by the LigBuilder program.

[0148]Once the active site has been graphically defined, the spatial and
electronic representations of a MarR modulating compound candidate can be
fit or docked within the target active site. Specific modifications of an
initial candidate can be made electronically, and then tested to
determine whether the complementarity between the active site and the
modulating compound candidate has been increased. To demonstrate the use
of the crystal structure for docking, the coordinates of the salicylate
were artificially removed from the MarR active site. Using this newly
created empty active site as input, the program FLEXX is able to predict
the proper binding orientation of salicylate with MarR (FLEXX Module, in
SYBYL. Tripos, Inc. 1699 Hanley Rd. St. Louis, Mo. 63144. Rarey, M. et
al. J. Mol. Bio. 1996, 261, 470-489). The result of this docking
experiment can be compared to the original salicylate orientation as
determined crystallographically. The dominant molecular interactions
between the salicylate and the active site residues may be predicted by
the docking algorithm, e.g. the carboxylate and hydroxyl groups.

Example 5

Use of Rational Drug Design to Identify MarR Modulating Compounds

[0149]One method of rational drug design techniques includes de novo drug
design which utilizes the structure of the protein to generate molecules
to dock within the active site. In this approach, a "seed" atom, or
seed-molecule with pre-defined attachment points is placed within the
active site. Programs are available to systematically "grow" chemical
modifications at the attachment points resulting in novel molecules.
Through an iterative process of growing and assessing the complimentarily
of the new structures, productive attachments can be saved, while
unproductive attachments are discarded. Subsequent redefinition of the
seed based on productive attachments can produce large number of drug
candidates for the specified target. This is an unbiased approach since
the result is not taken from a pre-existing virtual library, and is often
used to generate compounds that would otherwise not be considered based
on current proprietary knowledge or chemist's intuition.

[0150]This approach was applied to the MarR protein using the program
LigBuilder to produce a list of novel potential drug candidates.

EQUIVALENTS

[0151]Those skilled in the art will recognize, or be able to ascertain
using no more than routine experimentation, many equivalents to the
specific embodiments and methods described herein. Such equivalents are
intended to be encompassed by the scope of the following claims.